Design:

There are two possible ways to control the system. One of them is from the outside of the cell via two synthetic signal pathways leading to the production of either an ON or an OFF free gRNA. In these synthetic signal pathways, a signal molecule binds to a receptor, which then leads to the production of the specific gRNA. As signal molecules, we chose copper, ethanol and estradiol. The reason we chose these compounds is because they are already existing receptor systems (1, 2, 3) and these compounds are rather easy to come by. Copper for example is used as a pesticide in organic farming and ethanol is cheap and easily accessible. Also, the amount of signal molecules needed to induce the toggle switch should be very small, in theory (1).

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ill.1: Ethanol-inducible gene expression for application in plants (1)

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       <img src="" alt="EtOH Yeast construct">
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ill. 2: Ethanol-inducible gene expression modified for application in yeast. In comparison to ill.1 we only used different promoters and terminators.

Binding of ethanol to alcR (transcription factor) induces a conformational change which enables the transcription factor to bind to AlcA (promoter) and leads to the expression of our reporter gene (1) or if we want to use it in combination with the toggle switch to the expression of guide-RNA.

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ill. 3: Copper inducible gene expression for application in plants (3)

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   <img src="" alt="Estradiol Yeast construct">
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ill.4: Copper inducible gene expression modified for application in yeast In comparison to ill.3 we only used different promoters and terminators.

The yeast activation copper-metallothionein expression gene ACE1 encodes for a transcription factor which is regulated by copper. This transcription factor is able to bind to MRE, a chimeric promoter, which regulates the reporter gene (3) or if we want to use it in combination with the toggle switch the guide-RNA.

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ill.5: Estradiol inducible gene expression for application in plants (2)

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   <img src="" alt="Estradiol Yeast construct">
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ill.6: Estradiol inducible gene expression modified for application in yeast In comparison to ill.5 we only used different promoters and terminators.

If Estradiol binds to the XVE fusion protein the LexA operator fused with the minimal promoter sequence p35S -46 is activated, thus the gene expression of the reporter gene is induced (2) or if we want to use it in combination with the toggle switch the expression of guide-RNA.

The other way to induce a switch, which we came up with later in the project, is with Liposomes. In this case, the ON or OFF gRNA is bound and directly transported into the cell. The reason we chose to introduce a second way to control our toggle switch is that in case one of the receptors doesn’t work at the end of the lab phase, we can still control the toggle switch directly with the gRNA contained in the liposomes, without being dependent on a working receptor. Furthermore, we became aware after a meeting with a plant specialist at our university, that both ethanol as well as estradiol influence the physiology of plants when they are used to induce genes.

The toggle switch itself is based on a dCas9 system. It relies on dCas9, in complex with a specific guide-RNA, binding to to the respective site in the promoter of a gene and thereby blocking its transcription. In case of an ON signal, either delivered by liposomes or by a receptor pathway, ON free gRNA will be present in the cell, bind to dCas9 and direct it to its target sites in the regulatory region of our OFF gene and thereby block the expression. While the OFF gene is blocked, the ON genes can be translated and be expressed. Therefore ON gRNA can be produced and GFP, our reporter gene, can be expressed. This constant stream of new free ON gRNA will deny the expression of free OFF gRNA even after the original ON signal decays and will, therefore, hold the system in the ON state. The system will only switch into the OFF state if an external OFF signal produces an OFF free gRNA which binds to the target sites in the ON gene promoter region and thereby stops the constant flow of free ON gRNA and replaces it with its own gene product.

As a reporter-system, we use a special variety of GFP which has a very short half-life due to an ubiquitin-tag. This is important because in order to prove that the system can be started and shut off again, the fluorescence of the GFP has to vanish as soon as the OFF signal is given (4). If we would use a variety of GFP without ubiquitin, the fluorescence would decay only very slowly and we would have to wait hours or days to prove that the toggle switch is switched OFF.

A key part of our wet lab work was to create Vectors containing our designed genetic parts. We used the cloning technique called “Golden Gate”[5] to produce all the working constructs. It enabled us to clone multiple parts sequence optimized into specific golden-gate ready

The chassis for our system are yeast (Pichia pastoris CBS7435) and Arabidopsis thaliana. We chose Arabidopsis thaliana because its life cycle is very short for a plant (about 6 weeks from germination to mature seeds) and its genome has been fully sequenced. Another advantage is that there is a research group in our building that works a lot with Arabidopsis thaliana and who helped us a lot with their experience.

(1) Roslan, H. A., Salter, M. G., Wood, C. D., White, M. R. H., Croft, K. P., Robson, F., … Caddick, M. X. (2001). Characterization of the ethanol-inducible alc gene- expression system in Arabidopsis thaliana, 28.

(2) Zuo, J., Niu, Q., & Chua, N. (2000). An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants, 24, 265–273.

(3) Saijo, T., & Nagasawa, A. (2014). Development of a tightly regulated and highly responsive copper- inducible gene expression system and its application to control of flowering time, 47–59. <a href="https://doi.org/10.1007/s00299-013-1511-5">https://doi.org/10.1007/s00299-013-1511-5</a>

(4) Houser, John R.; Ford, Eintou; Chatterjea, Sudeshna M.; Maleri, Seth; Elston, Timothy C.; Errede, Beverly (2012): An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. In Yeast (Chichester, England) 29 (12), pp. 519–530. DOI: 10.1002/yea.2932.

(5) Engler, Carola; Marillonnet, Sylvestre (2014): Golden Gate cloning. In Methods in molecular biology (Clifton, N.J.) 1116, pp. 119–131. DOI: 10.1007/978-1-62703-764-8_9.

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Design: